B7-h3 antibody, antigen-binding fragment thereof and medical use thereof

ABSTRACT

A B7-H3 antibody, an antigen-binding fragment thereof and a medical use thereof are provided. Furthermore, a pharmaceutical composition containing the B7-H3 antibody or antigen-binding fragment thereof, and the use thereof as a medicament are provided. In particular, a use of a human B7-H3 antibody or antigen-binding fragment thereof for the manufacture of a medicament for the treatment of a B7-H3-associated disease or condition are described.

FIELD OF THE INVENTION

The present invention relates to a B7-H3 antibody or antigen-bindingfragment thereof. The present invention further relates to a humanantibody comprising the CDR regions of the B7-H3 antibody. The presentinvention also relates to a pharmaceutical composition comprising theB7-H3 antibody or antigen-binding fragment thereof, and its use as adiagnostic reagent and therapeutic agent for B7-H3 related diseases.

BACKGROUND OF THE INVENTION

The T cell-mediated immune response plays an extremely important role inanti-tumor processes of an organism, however, the activation andproliferation of T cells requires not only an antigen signal recognizedby the T cell receptor (TCR), but also a second signal provided byco-stimulatory molecules. The molecules of the B7 family belong to theco-stimulatory molecule immunoglobulin superfamily. More and morestudies have shown that molecules of this family play an importantregulatory role in the normal immune function and pathological state inan organism.

B7-H3 is a member of B7 family and is a type I transmembrane protein,which contains a signal peptide at the amino terminus, an extracellularimmunoglobulin-like variable region (IgV) and constant region (IgC), atransmembrane region, and a cytoplasmic tail region having 45 aminoacids (Tissue Antigens. 2007 August; 70(2): 96-104). B7-H3 has two kindsof splicing variants, B7-H3a and B7-H3b. The extracellular domain ofB7-H3a consists of two immunoglobulin domains of IgV-IgC (also known as2IgB7-H3), while the extracellular domain of B7-H3b consists of fourimmunoglobulin domains of IgV-IgC-IgV-IgC (also known as 4IgB7-H3).

B7-H3 protein is not expressed or is poorly expressed in normal tissuesand cells, but highly expressed in various tumor tissues and is closelycorrelated with tumor progression, patient survival and prognosis. Ithas been clinically reported that B7-H3 is over-expressed in many typesof cancers, especially in non-small cell lung cancer, renal cancer,urinary tract epithelial cancer, colorectal cancer, prostate cancer,glioblastoma multiforme, ovarian cancer and pancreas cancer (LungCancer. 2009 November; 66(2): 245-249; Clin Cancer Res. 2008 Aug. 15;14(16): 5150-5157). In addition, it has also been reported in theliterature that, in prostate cancer, the expression level of B7-H3 ispositively correlated with clinical pathological malignancy (such astumor volume, extra-prostatic invasion or Gleason score), and is alsoassociated with cancer progression (Cancer Res. 2007 Aug. 15;67(16):7893-7900). Similarly, in glioblastoma multiforme, the expressionof B7-H3 is inversely associated with event-free survival, and inpancreatic cancer, the expression of B7-H3 is associated with lymph nodemetastasis and pathological progression. Therefore, B7-H3 is consideredas a new tumor marker and potential therapeutic target.

Currently, there have been therapeutic strategies specific for B7-H3target for preclinical studies. For example, antibodies targeting murineB7-H3 will enhance infiltrative CD8-positive T cells in tumors andinhibit tumor growth (Mod Pathol. 2010 August; 23(8): 1104-1112).Furthermore, patent WO 2008/066691 shows that antibodies recognizing theB7-H3 variant, B7-H3a, exhibited an in vivo anti-tumor effect onadenocarcinoma. In clinical studies, an ADC of murine B7-H3 antibodyconjugated with radioactive I¹³¹ significantly inhibited the growth ofneuroblastoma in patients (J Neufooocol 97(3):409-18 (2010)). However,the B7H3 antibodies currently under study are humanized antibodies thathave been engineered by humanization of murine antibodies. However,humanized antibodies upon immunization have higher immunogenicity thanfully human antibodies which do not contain any murine antibodycomponents. This higher immunogenicity is an unfavorable factor in humanapplication.

Phage display technology refers to the fusion of an exogenous protein orpolypeptide with a phage coat protein, so as to express an exogenousprotein on the surface of the phage. The phage antibody library is anantibody library established by combining phage display technology, PCRamplification technology and protein expression technology.

The biggest advantage of the phage antibody library is to prepare thefully human antibody by mimicking the three processes of antibodyproduction in vivo without immunization in vivo. In addition, the phageantibody library has the following advantages: 1) The unification ofgenotype and phenotype is achieved. In addition, the experimental methodis simple and rapid. Whereas the traditional antibody production methodby hybridoma technology takes several months, the antibody librarytechnology takes only a few weeks. 2) The expressed product is a fullyhuman antibody, and the molecular weight thereof is small. The antibodyis mainly expressed in the form of active fragments Fab and scFv andwhen compared with complete antibody, it has obvious advantages intissue penetrability. 3) Screening capacity is large: hybridomatechnology is used to screen among thousands of clones, but antibodylibrary technology can be used to select from millions or even hundredsof millions of molecules; therefore, more types of antibodies will beobtained. 4) Wide application: utilizing prokaryotic expression systemsleads to more obvious advantage in large scale production (Curr OpinBiotechnol. 2002 December; 13(6):598-602; Immunotechnology, 2013, 48(13)48(13): 63-73).

At present, patents such as WO2008100934, WO2010096734, WO2012147713,WO2015181267, WO2016044383 etc. have reported B7-H3 antibodies, however,most of them are murine antibodies or humanized antibodies. Most ofthese antibodies are still in clinical phase I and discovery phase,either in domestic or overseas, and none of antibody drug targetingB7-H3 is available in market. Thus, it is necessary to further develop aB7-H3 fully human antibody with higher activity, high affinity and highstability, for the treatment of related diseases and application.

SUMMARY OF THE INVENTION

One purpose of the present invention is to provide a monoclonal antibodyor antigen-binding fragment thereof that binds to the amino acidsequence(s) or three-dimensional structure of the B7-H3 extracellularregion. Furthermore, another purpose of the present invention is toscreen and obtain highly active and highly stable anti-human B7-H3 fullyhuman antibodies that compete with the monoclonal antibody or antibodyfragments thereof.

Furthermore, the present invention provides a DNA encoding the antibody,a vector comprising the DNA, a transformant obtained by transforming thevector, a method of producing an antibody or antibody fragment thereofusing the transformant, and a diagnostic reagent or therapeutic agent inwhich said antibody or antibody fragment thereof is served as activeingredient.

In one aspect, the present invention provides a B7-H3 antibody orantigen-binding fragment thereof which binds to human B7-H3, wherein theB7-H3 antibody or antigen-binding fragment thereof is selected from anyone of the monoclonal antibodies or antigen-binding fragments thereof ofthe following (i) to (ii):

(i) a monoclonal antibody or antigen-binding fragment thereof,comprising one or more CDR region sequences selected from the followingsequences or selected from the amino acid sequences with at least 95%identity to the following:

antibody heavy chain variable region HCDR sequences as shown in SEQ IDNO: 10, 11 and 12; and antibody light chain variable region LCDRsequences as shown in SEQ ID NO: 13, 14 and 15;

(ii) a monoclonal antibody or antigen-binding fragment thereof,comprising one or more CDR region sequences selected from the followingsequences or selected from the amino acid sequences with at least 95%identity to the following:

antibody heavy chain variable region HCDR sequences as shown in SEQ IDNO: 16, 17 and 18; and antibody light chain variable region LCDRsequences as shown in SEQ ID NO: 19, 20 and 21.

In a preferred embodiment, a B7-H3 antibody or antigen-binding fragmentthereof according to the present invention is provided, wherein themonoclonal antibody is a recombinant antibody.

In a preferred embodiment, a B7-H3 antibody or antigen-binding fragmentthereof according to the present invention is provided, wherein themonoclonal antibody is a human recombinant antibody or antigen-bindingfragment thereof.

In a preferred embodiment, the B7-H3 antibody or antigen-bindingfragment thereof according to the present invention is provided, whereinthe framework (FR) sequences of light chain and the heavy chain variableregions of human recombinant antibody are derived from a human germlinelight chain and heavy chain, respectively, or mutant sequence thereof.

In a preferred embodiment, a B7-H3 antibody or antigen-binding fragmentthereof according to the present invention is provided, wherein thehuman recombinant antibody comprises a heavy chain variable region asshown in SEQ ID NO: 6 or 8 or a variant thereof; wherein the variant has1-10 amino acid substitution(s) in the heavy chain variable regionsequence as shown in SEQ ID NO: 6 or 8.

In a preferred embodiment, a B7-H3 antibody or antigen-binding fragmentthereof according to the present invention is provided, wherein thehuman recombinant antibody comprises a light chain variable region asshown in SEQ ID NO: 7 or 9 or a variant thereof; wherein the variant has1-10 amino acid substitution(s) in the light chain variable regionsequence of SEQ ID NO: 7 or 9.

In a preferred embodiment, a B7-H3 antibody or antigen-binding fragmentthereof according to the present invention is provided, wherein theB7-H3 antibody further comprises a human antibody constant region,preferably the B7-H3 antibody is a full-length antibody consisting ofthe heavy chain and light chain sequence as shown in SEQ ID NO: 22 and23, respectively; or a full-length antibody consisting of the heavychain and light chain sequence as shown in SEQ ID NO: 22 and 26,respectively; or a full-length antibody consisting of the heavy chainand light chain sequence as shown in SEQ ID NO: 24 and 25, respectively.

In a preferred embodiment, a B7-H3 antibody or antigen-binding fragmentthereof according to the present invention is provided, wherein theantigen-binding fragment is selected from the group consisting of Fab,Fab′, F(ab′)2, single-chain antibody (scFv), dimerized V region(diabody), disulfide-stabilized V region (dsFv) and CDR-containingpeptide.

In another aspect, the present invention provides an isolated B7-H3antibody or antigen-binding fragment thereof, characterized in that itcompetes with the B7-H3 antibody or antigen-binding fragment thereof asdescribed above for binding to human B7-H3.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of the B7-H3 antibody orantigen-binding fragment thereof according to the present invention, andone or more pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a nucleic acid molecule encoding theB7-H3 antibody or antigen-binding fragment thereof as described in thepresent invention.

The present invention also provides a recombinant vector comprising thenucleic acid molecule as described above.

The present invention also provides a host cell transformed with therecombinant vector as described above, the host cell is selected fromthe group consisting of prokaryotic cell and eukaryotic cell, preferablyeukaryotic cell, more preferably mammalian cell or yeast cell.

The present invention also provides a method for producing the antibodyor antigen-binding fragment thereof according to present invention,wherein the method includes culturing the host cell as described abovein a culture to form and accumulate the B7-H3 antibody orantigen-binding fragment thereof according to present invention, andrecovering the accumulated antibody or antigen-binding fragment thereoffrom the culture.

The present invention also provides a method for immunologicallydetecting or measuring B7-H3, wherein the method comprises utilizing theB7-H3 antibody or antigen-binding fragment thereof as described in thepresent invention.

The present invention also provides a reagent for detecting or measuringhuman B7-H3, wherein the reagent comprises the B7-H3 antibody orantigen-binding fragment thereof as described in the present invention.

The present invention also provides a diagnostic reagent for detectingdiseases associated with B7-H3 positive cells, the diagnostic reagentcomprises the B7-H3 antibody or antigen-binding fragment thereof asdescribed in the present invention.

The present invention also provides a method for diagnosing diseasesrelated to B7-H3 positive cells, the method comprises detecting ordetermining B7-H3 or B7-H3 positive cells by using the B7-H3 antibody orantigen-binding fragment thereof as described in the present invention.

In another aspect, the present invention also provides the use of theB7-H3 antibody or antigen-binding fragment thereof as described in thepresent invention for the preparation of a diagnostic reagent fordiseases related to B7-H3 positive cells.

In another aspect, the present invention further provides a therapeuticagent for treating diseases associated with B7-H3 positive cells, thetherapeutic agent comprises the B7-H3 antibody or antigen-bindingfragment thereof as described in the present invention, or comprises thepharmaceutical composition as described above, or the nucleic acidmolecule as described above.

The present invention also provides a method for treating diseasesrelated to B7-H3 positive cells, the method comprises inducing celldeath of B7-H3 positive cells by utilizing the antibody orantigen-binding fragment thereof as described in the present invention,or the pharmaceutical composition as described above, or the nucleicacid molecule as described above.

In another aspect, the present invention further provides the use of theantibody or antigen-binding fragment thereof, or the pharmaceuticalcomposition, or the nucleic acid molecule as described in the presentinvention in the preparation of therapeutic agents for treating diseasesrelated to B7-H3 positive cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Binding ability of different antibodies to human 2Ig-B7-H3antigen;

FIG. 2: Binding ability of different antibodies to human 4Ig-B7-H3antigen;

FIG. 3: Cross-binding ability of different antibodies to murine B7-H3antigen;

FIG. 4: Binding ability of different antibodies to U87MG cells;

FIG. 5: Endocytic effect of different antibodies on U87MG cells.

DETAILED DESCRIPTION OF THE INVENTION 1. Terms

In order to more readily understand the present invention, certaintechnical and scientific terms are specifically defined below. Unlessspecifically indicated elsewhere in this document, all other technicaland scientific terms used herein have the meaning commonly understood byone of ordinary skill in the art to which this invention pertains.

As used herein, the three-letter code and the single-letter code foramino acids are as described in J. biol. chem, 243, p 3558 (1968).

As used herein, “antibody” refers to immunoglobulin, a structure offour-peptide chains connected together by disulfide bonds between twoidentical heavy chains and two identical light chains. Differentimmunoglobulin heavy chain constant regions exhibit different amino acidcompositions and rank orders, hence present different kinds ofantigenicity. Accordingly, immunoglobulin can be divided into fivecategories, or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA andIgE, with heavy chain μ, δ, γ, α and ε, respectively. According to itsamino acid composition of the hinge region and the number and locationof heavy chain disulfide bonds, the same type of Ig can be divided intodifferent sub-categories. For example, IgG can be divided into IgG1,IgG2, IgG3, and IgG4. Light chains can be divided into κ or λ chain, dueto different constant regions. Each IgG among the five types has κ or λchain.

In the present invention, the antibody light chain as described hereinfurther comprises a light chain constant region, wherein the lightconstant region comprises a human or murine κ, λ chain or a variantthereof.

In the present invention, the antibody heavy chain as described hereinfurther comprises a heavy chain constant region, wherein the heavy chainconstant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or avariant thereof.

The sequence of about 110 amino acids closest to the N-terminus of theantibody heavy and light chains, changes largely, known as the variableregion (Fv region). The sequence of amino acids closest to theC-terminus is relatively stable, known as constant region. The variableregion comprises three hypervariable regions (HVR) and four frameworkregions (FR) having relatively conserved sequences. Three hypervariableregions determine the specificity of the antibody, also known ascomplementarity determining regions (CDRs). Each light chain variableregion (LCVR) and each heavy chain variable region (HCVR) comprisesthree CDR regions and four FR regions. Sequentially ordered from theamino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The three light chain CDRs are referred to as LCDR1,LCDR2, and LCDR3. The three heavy chain CDRs are referred to as HCDR1,HCDR2 and HCDR3. The number and location of the CDR amino acid residuesin the LCVR and HCVR regions of the antibody or antigen binding fragmentherein comply with known Kabat numbering criteria (e.g., LCDR1-3,HCDR2-3), or comply with Kabat and Chothia numbering criteria (e.g.,HCDR1).

The terms “human antibody” and “human derived antibody” are usedinterchangeably and refer to an antibody comprising one or more variableand constant regions derived from a human immunoglobulin sequence. In apreferred embodiment of the invention, all of the variable and constantregions are derived from human immunoglobulin sequences, i.e., “fullyhuman derived antibody” or “fully human antibody”. These antibodies canbe obtained in a variety of ways, including antibodies obtained by usingphage display technology, including isolating B cells from human PBMC,spleen, lymph node tissue and constructing natural single-stranded phagehuman antibody library, or by immunizing transgenic mice expressinghuman antibody light and heavy chain and screening.

The term “murine antibody” used in the present invention refers to amonoclonal antibody against human B7-H3 prepared according to theknowledge and skill in the art. During preparation, the test subject isinjected with a B7-H3 antigen, and then the hybridoma expressingantibodies having desired sequences or functional properties areisolated. In a preferred embodiment of the invention, the murine B7-H3antibody or antigen-binding fragment thereof further comprises a lightchain constant region of a murine kappa, lambda chain or a variantthereof, or further comprises a heavy chain constant region of murineIgG1, IgG2, IgG3 or variants thereof.

The term “chimeric antibody” is an antibody which is formed by fusingthe variable region of a murine antibody with the constant region of ahuman antibody, so as to alleviate the murine antibody-induced immuneresponse. To establish a chimeric antibody, a hybridoma secreting aspecific murine monoclonal antibody is established and a variable regiongene is cloned from the murine hybridoma cells. Then a desired constantregion gene of a human antibody is cloned and connected with the murinevariable region genes to form a chimeric gene which can be subsequentlyinserted into an expression vector. Finally the chimeric antibodymolecule is expressed in eukaryotic or prokaryotic system. In apreferred embodiment of the present invention, the light chain of theB7-H3 chimeric antibody further comprises a light chain constant regionderived from the human kappa, lambda chain or a variant thereof. Theheavy chain of the B7-H3 chimeric antibody further comprises a heavychain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or avariant thereof, and preferably comprises a heavy chain constant regionderived from human IgG1, IgG2 or IgG4, or variant of IgG1, IgG2 or IgG4with amino acid mutations (such as YTE mutation or back mutation).

The term “humanized antibody”, also known as CDR-grafted antibody,refers to an antibody generated by grafting murine CDR sequences into avariable region framework of a human antibody (i.e. antibodies producedwithin different types of human germline antibody framework sequences).A humanized antibody overcomes the heterologous response induced by achimeric antibody that carries a large amount of murine proteincomponents. Such framework sequences can be obtained from public DNAdatabases including germline antibody gene sequences or publishedreferences. For example germline DNA sequences of human heavy and lightchain variable region genes can be found in e.g., “VBase” human germlinesequence database (available on the Internet atwww.mrccpe.com.ac.uk/vbase), as well as found in Kabat, E A, et al, 1991Sequences of Proteins of Immunological Interest, 5th edition.

The CDR graft can reduce the affinity of the B7-H3 antibody orantigen-binding fragment thereof to the antigen, due to the frameworkresidues that are in contact with the antigen. Such interaction can bethe result of hyper-mutation in somatic cells. Therefore, it may stillbe necessary to graft such donor framework amino acids onto theframework of humanized antibodies. Amino acid residues from a non-humanB7-H3 antibody or antigen-binding fragment thereof which are involved inantigen binding can be identified by examining the murine monoclonalantibody variable region sequences and structures. Each residue in theCDR donor framework that differs from the germline can be considered tobe relevant. If the closest germline cannot be determined, the sequencecan be compared with the common sequence of a subtype or the sequence ofthe murine with a high similarity percentage. Rare framework residuesare thought to be the result of somatic hyper-mutation and thus play animportant role in binding.

The term “antigen-binding fragment” or “functional fragment” of anantibody refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., B7-H3). It has beenshown that fragments of full-length antibodies can be used for theantigen-binding function of antibodies. Examples of the bindingfragments included in the term “antigen-binding fragment” of an antibodyinclude: (i) Fab fragment, a monovalent fragment consisting of VL, VH,CL and CH1 domains; (ii) F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by disulfide bonds in the hingeregion, (iii) Fd fragment, consisting of the VH and CH1 domains; (iv) Fvfragment, consisting of the VH and VL domains of one arm of theantibody; (v) single domain or dAb fragment (Ward et al. (1989) Nature341: 544-546) composed of the VH domain; and (vi) a separatecomplementarity determining region (CDR); or (vii) a combination of twoor more separated CDRs optionally linked by a synthetic linker.Furthermore, although the VL domain and VH domain of the Fv fragment areencoded by separate genes, they can be linked by a synthetic linkerusing recombinant methods such that they can generate a single proteinchain with monovalent molecular structure by pairing the VL and VHdomains (called single-chain Fv (scFv); see, e.g., Bird et al. (1988)Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA85: 5879-5883). Such single chain antibodies are also intended to beincluded in the term “antigen-binding fragment” of an antibody. Suchantibody fragments are obtained using conventional techniques known inthe art, and functional screening of fragments are used in the same wayas the intact antibodies. The antigen binding sites can be produced byrecombinant DNA techniques or by enzymatic or chemical disruption of theintact immunoglobulin. The antibodies may be in different phenotype,e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgEor IgM antibody.

The antigen-binding fragments of the present invention include Fab,F(ab′)2, Fab′, single-chain antibody (scFv), dimerized V region(diabody), disulfide-stabilized V region (dsFv), CDR-containing peptide,etc.

Fab is an antibody fragment having a molecular weight of about 50,000and having antigen-binding activity, such fragments are obtained bytreating an IgG antibody molecule with protease papain (cleaving aminoacid residue at position 224 of H chain), wherein about half of theN-terminal side of the H chain and the entire L chain are bound bydisulfide bond.

The Fab of the present invention can be produced by treating themonoclonal antibodies of the present invention, which specificallyrecognizes human B7-H3 and binds to the extracellular region amino acidsequence or three-dimensional structure thereof, with papain.Furthermore, the Fab can be produced by inserting a DNA encoding Fab ofthe antibody into a prokaryotic expression vector or eukaryoticexpression vector and introducing the vector into prokaryote oreukaryote to express the Fab.

F(ab′)2 is an antibody fragment obtained by digesting the lower part oftwo disulfide bonds in IgG hinge region with pepsin. It has a molecularweight of about 100,000 and antigen-binding activity, and comprises twoFab regions linked at the hinge position.

The F(ab′)2 of the present invention can be produced by treating themonoclonal antibody of the present invention, which specificallyrecognizes human B7-H3 and binds to the extracellular region amino acidsequence or three-dimensional structure thereof, with pepsin.Furthermore, the F(ab′)2 can be produced by linking the Fab′ describedbelow with a thioether bond or a disulfide bond.

Fab′ is an antibody fragment having a molecular weight of about 50,000and having antigen-binding activity. It is obtained by cleaving thedisulfide bond in the hinge region of the F(ab′)2 mentioned above. TheFab′ of the present invention may be produced by treating the F(ab′)2 ofthe present invention, which specifically recognizes human B7-H3 andbinds to the extracellular region amino acid sequence orthree-dimensional structure thereof, with a reducing agent (such asdithiothreitol).

Furthermore, the Fab′ can be produced by inserting a DNA encoding a Fab′fragment of the antibody into a prokaryotic expression vector or aeukaryotic expression vector and introducing the vector into prokaryoteor eukaryote to express the Fab′.

The term “single-chain antibody”, “single-chain Fv” or “scFv” refers toa molecule comprising an antibody heavy chain variable domain (orregion; VH) and an antibody light chain variable domain (or region; VL)connected by a linker. Such scFv molecules have the general structure:NH₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable linkers inprior art consist of repeated GGGGS amino acid sequence or variantsthereof, for example a variant having 1-4 repeats (Holliger et al.(1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers thatcan be used in the present invention are described in Alfthan et al.(1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol.31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov etal. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), CancerImmunol.

The scFv of the present invention can be produced by the followingsteps: obtaining the cDNA encoding VH and VL of the monoclonal antibodyof the present invention which specifically recognizes human B7-H3 andbinds to the extracellular region amino acid sequence orthree-dimensional structure thereof; constructing a DNA encoding thescFv; inserting the DNA into a prokaryotic expression vector or aeukaryotic expression vector; and then introducing the expression vectorinto prokaryote or eukaryote to express said scFv.

A diabody is an antibody fragment in which the scFv is dimerized, and isan antibody fragment having bivalent antigen-binding activity. In thebivalent antigen binding activity, the two antigens may be the same ordifferent.

The diabody of the present invention can be produced by the followingsteps: obtaining the cDNA encoding VH and VL of the monoclonal antibodyof the present invention which specifically recognizes human B7-H3 andbinds to the extracellular region amino acid sequence orthree-dimensional structure thereof; constructing a DNA encoding scFvsuch that the length of the linker peptide is 8 or less amino acidresidues; inserting the DNA into a prokaryotic expression vector or aeukaryotic expression vector; and then introducing the expression vectorinto prokaryote or eukaryote to express the diabody.

The dsFv is obtained by substituting one amino acid residue in each ofthe VH and the VL with cysteine residue, and then linking thepolypeptides via disulfide bond between the two cysteine residues. Theamino acid residue to be substituted with a cysteine residue can beselected based on a three-dimensional structure prediction of theantibody in accordance with known methods (Protein Engineering, 7, 697(1994)).

The dsFv of the present invention can be produced by the followingsteps: obtaining the cDNA encoding the VH and the VL of the monoclonalantibody of the present invention which specifically recognizes humanB7-H3 and binds to the extracellular region amino acid sequence orthree-dimensional structure thereof; constructing a dsFv-encoding DNA;inserting the DNA into prokaryotic expression vector or eukaryoticexpression vector; and then introducing the expression vector intoprokaryote or eukaryote to express said dsFv.

The CDR-containing peptide is constructed by one or more regions of CDRsof VH or VL. Peptides comprising several CDRs can be linked directly orvia a suitable peptide linker.

The CDR-containing peptide of the present invention can be produced bythe following steps: constructing a DNA encoding CDRs of the VH and theVL of the monoclonal antibody of the present invention whichspecifically recognizes human B7-H3 and binds to the extracellularregion amino acid sequence or three-dimensional structure thereof;inserting the DNA into a prokaryotic expression vector or a eukaryoticexpression vector; and then introducing the expression vector intoprokaryote or eukaryote to express said peptide. The CDR-containingpeptide can also be produced by chemical synthesis methods such as Fmocmethod or tBoc method.

The term “CDR” refers to one of the six hypervariable regions within thevariable domain of an antibody that primarily contributes to antigenbinding. One of the most commonly used definitions for the six CDRs isprovided by Kabat E. A. et al. (1991) Sequences of proteins ofimmunological interest. NIH Publication 91-3242. As used herein, theKabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the lightchain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), as well asCDR2 and CDR3 of heavy chain variable domain (CDR H2, CDR H3 or H2, H3).

The term “antibody framework” as used herein, refers to a portion of thevariable domain VL or VH, which serves as a scaffold for the antigenbinding loop (CDR) of the variable domain. Essentially, it is a variabledomain without CDRs.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds (e.g.,a specific site on B7-H3 molecule). Epitopes typically include at least3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous ornon-contiguous amino acids in a unique spatial conformation. See, forexample, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996).

The terms “specific binding”, “selective binding”, “selectively bind”and “specifically bind” refer to the binding of an antibody to anepitope on a predetermined antigen. Typically, the antibody binds withan affinity (KD) of less than about 10⁻⁷ M, such as approximately lessthan about 10⁻⁸ M, 10⁻⁹M or 10⁻¹⁰M or less.

The term “KD” or “Kd” refers to the dissociation equilibrium constantfor a particular antibody-antigen interaction. Typically, the antibodyof the present invention binds to B7-H3 with a dissociation equilibriumconstant (KD) of less than about 10⁻⁷ M, such as less than about 10⁻⁸ M,10⁻⁹ M or 10⁻¹⁰ M or less, for example, as determined using surfaceplasmon resonance (SPR) techniques in a BIACORE instrument.

The term “competitive binding” refers to an antibody that recognizes andbinds to the same epitope (also known as antigenic determinant) or aportion thereof of the extracellular domain of human B7H3 as the onerecognized by monoclonal antibody of the present invention. An antibodythat binds to the same epitope as the monoclonal antibody of the presentinvention refers to an antibody that recognizes and binds to the aminoacid sequence of human B7-H3 recognized by the monoclonal antibody ofthe present invention.

The term “nucleic acid molecule” as used herein refers to a DNA moleculeand a RNA molecule. The nucleic acid molecule may be single stranded ordouble stranded, but is preferably a double stranded DNA. A nucleic acidis “effectively linked” when it is placed into functional relationshipwith another nucleic acid sequence. For example, if a promoter orenhancer affects transcription of a coding sequence, the promoter orenhancer is effectively linked to the coding sequence.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. In oneembodiment, the vector is a “plasmid” which refers to a circular doublestranded DNA loop into which additional DNA segment can be ligated. Inanother embodiment, the vector is a viral vector, wherein an additionalDNA segment can be ligated into viral genome. The vectors disclosedherein are capable of self-replicating in a host cell into which theyhave been introduced (for example, a bacterial vector having a bacterialreplication origin and a episomal mammalian vector) or can be integratedinto the genome of a host cell upon introduction into host cell, therebyis replicated along with the host genome (e.g., a non-episomal mammalianvector).

Methods for producing and purifying antibodies and antigen-bindingfragments are well known in the art, such as Cold Spring Harbor AntibodyTechnical Guide, Chapters 5-8 and 15. For example, mice can be immunizedwith human B7-H3 or a fragment thereof, and the obtained antibody can bere-natured, purified, and sequenced by using conventional methods knownin the art. The antigen-binding fragment can also be prepared byconventional methods. The antibodies or antigen-binding fragments of theinvention are genetically engineered to add one or more human FR regionsin non-human CDR regions. The human FR germline sequence(s) can beobtained by aligning human antibody variable germlines in gene databasesand MOE software from the ImMunoGeneTics (IMGT) website athttp://imgt.cines.fr or from the Journal of Immunoglobulins20011SBN012441351.

The term “host cell” refers to a cell into which an expression vectorhas been introduced. Host cells can include bacterial, microbial, plantor animal cells. Bacteria susceptible to be transformed include membersof the Enterobacteriaceae family, such as strains of Escherichia coli orSalmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus;Streptococcus and Haemophilus influenzae. Suitable microorganismsinclude Saccharomyces cerevisiae and Pichia pastoris. Suitable animalhost cell lines include CHO (Chinese hamster ovary cell line) and NS0cells.

The engineered antibody or antigen-binding fragment of the presentinvention can be prepared and purified by conventional methods. Forexample, cDNA sequence(s) encoding a heavy chain and a light chain canbe cloned and recombined into a GS expression vector. The recombinantimmunoglobulin expression vector can be stably transfected in CHO cells.Mammalian expression systems result in glycosylation of antibodies,particularly at the highly conserved N-terminal site in the Fc region.Stable clones are obtained by expressing antibodies that specificallybind to human B7-H3. Positive clones are expanded in serum-free mediumin a bioreactor to produce antibodies. The culture medium containing thesecreted antibody can be purified by conventional technique. Forexample, purification is carried out using an A or G Sepharose FF columnthat has been equilibrated with a compatible buffer. Thenon-specifically bound components are removed by washing. The boundantibody is eluted by a pH gradient method, and the antibody fragmentsare detected by SDS-PAGE and collected. The antibody can be filtered andconcentrated by a conventional manner. Soluble aggregate and multimerscan also be removed by conventional methods such as size exclusion orion exchange. The product needs to be frozen immediately, such as at−70° C., or lyophilized.

“Administration” and “treatment”, when applied to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refer tocontact with an exogenous pharmaceutical, therapeutic, diagnosticreagent, or composition with the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contacting thecell with a reagent, as well as contacting a fluid with a reagent,wherein the fluid is in contact with the cell. “Administration” and“treatment” also mean in vitro and ex vivo treatments, e.g., of a cell,by a reagent, diagnostic, binding composition or by another cell.“Treatment”, when applied to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,research and diagnostic applications.

“Treat” means to administer a therapeutic agent, such as a compositioncomprising any of the binding compounds of the present invention,internally or externally to a patient having one or more diseasesymptoms for which the agent has known therapeutic activity. Typically,the agent is administered in an amount effective to alleviate one ormore disease symptoms in the treated patient or population, so as toinduce the regression of such symptom(s) or to prevent the progressionby any clinically measurable degree. The amount of a therapeutic agentthat is effective to alleviate any particular disease symptom (alsoreferred to “therapeutically effective amount”) may vary according tofactors such as the disease state, age and weight of the patient, andthe ability of the agent to elicit desired response in the patient.Whether a disease symptom has been alleviated can be assessed by anyclinical measurement typically used by physicians or other skilledhealthcare providers to assess the severity or progression status ofthat symptom. Even if an embodiment of the present invention (e.g., atreatment method or article of manufacture) is not effective inalleviating the target disease symptom(s) in every patient, it shouldalleviate the target disease symptom(s) in a statistically significantnumber of patients as determined by any statistical test known in theart such as the Student's t-test, chi-square test, U-test according toMann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra testand Wilcoxon test.

“Conservative modification” or “conservative replacement orsubstitution” refers to substitutions of amino acids in a protein withother amino acid having similar characteristics (e.g., charge, sidechain size, hydrophobicity/hydrophilicity, backbone conformation andrigidity, etc.), such that the changes can be frequently made withoutaltering the biological activity of the protein. It will be appreciatedby those skilled in the art that, in general, a single amino acidsubstitution in a non-essential region of polypeptide does notsubstantially alter biological activity (see, for example, Watson et al.(1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co.,Page 224, (4th edition)). In addition, substitutions with structurallyor functionally similar amino acids are unlikely to disrupt biologicalactivity.

An “effective amount” includes an amount sufficient to ameliorate orprevent a symptom or condition of medical disease. An effective amountalso means an amount sufficient to allow or facilitate the diagnosis. Aneffective amount for a particular patient or veterinary subject can varydepending on factors such as: the condition to be treated, the overallhealth condition of the patient, the route and dose of administration,and the severity of side effects. An effective amount can be the maximumdose or dosing regimen that avoids significant side effects or toxiceffects.

“Exogenous” refers to a substance that is produced outside of organism,cell or human, depending on the situation. “Endogenous” refers to asubstance that is produced in a cell, organism or human, depending onthe situation.

“Identity” refers to the sequence similarity between two polynucleotidesequences or two polypeptide sequences. When the positions in twosequences to be compared are occupied by the same base or amino acidmonomer subunit, for example, if each position of two DNA molecules isboth occupied by adenine, the molecules are considered to be homologousat that position. The percent identity between the two sequences is afunction of the number of matched or homologous positions shared by twosequences divided by the number of positions to be compared×100. Forexample, in the optimal alignment of sequence(s), if there are 6 matchesor homologs among 10 positions between two sequences, then the twosequences are deemed as having 60% homology; if there are 95 matches orhomologs among 100 positions between two sequences, then the twosequences are deemed as having 95% homology. In general, a comparison isperformed when the maximum identity percentage is obtained by aligningtwo sequences.

As used herein, the expressions “cell”, “cell line” and “cell culture”are used interchangeably, and all such terms include the progenythereof. Thus, the words “transformant” and “transformed cell” includeprimary test cells and cultures derived therefrom, regardless of thenumber of passages. It should also be understood that all progeny maynot be exactly identical in terms of DNA content due to deliberate orinadvertent mutations. The mutant progeny having the same function orbiological activity as screened for the primarily transformed cell isincluded. In the case of a different name, it is clearly understood fromthe context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which small amount of particular portion ofnucleic acid, RNA, and/or DNA are amplified as described for example inU.S. Pat. No. 4,683,195. In general, it is necessary to obtain sequenceinformation from the end or beyond the target region, therebyoligonucleotide primers can be designed; these primers are identical orsimilar to the opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers may be identical to the ends ofthe material to be amplified. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, phage or plasmid sequences, etc.See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant.Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).The PCR used herein is considered as an example (but not the only one)of nucleic acid polymerase reaction method for amplifying a nucleic acidtest sample, said method comprises the use of known nucleic acids as aprimer and nucleic acid polymerase to amplify or produce a specificportion of the nucleic acid.

“Optional” or “optionally” means that the event or situation describedsubsequently may (but need not to) occur, this includes where the eventor situation occurs or does not occur. For example, “optionallycomprising 1-3 antibody heavy chain variable regions” means that theantibody heavy chain variable region with specific sequence can be, butneed not to, be present.

“Pharmaceutical composition” means a mixture comprising one or morecompounds described herein or a physiologically/pharmaceuticallyacceptable salt or prodrug thereof, along with other chemicalcomponents, such as physiological/pharmaceutically acceptable carrierand excipient. The purpose of the pharmaceutical composition is topromote the administration to the organism, and facilitate theabsorption of active ingredient and thereby exert a biological activity.

Furthermore, the present invention relates to a method forimmunologically detecting or measuring B7-H3, a reagent forimmunologically detecting or measuring B7-H3, a method forimmunologically detecting or measuring cells expressing B7-H3, and adiagnostic reagent for diagnosis of disease related to B7-H3 positivecells, comprising the monoclonal antibody or antibody fragment of thepresent invention that specifically recognizes human B7-H3 and binds toextracellular region amino acid sequence or three-dimensional structurethereof, as an active ingredient.

In the present invention, the method for detecting or determining theamount of B7-H3 may be any known method. For example, it includesimmunodetection or assay.

The immunodetection or assay is a method of detecting or determining theamount of antibody or antigen by using labeled antigen or antibody.Examples of immunodetection or assay include a radioactive substancelabeled immunological antibody method (RIA), an enzyme immunoassay (EIAor ELISA), a fluorescent immunoassay (FIA), a luminescent immunoassay, awestern blotting method, physicochemical methods, etc.

The above-mentioned diseases related to B7-H3 positive cells can bediagnosed by detecting or measuring cells expressing B7-H3 by using themonoclonal antibodies or antibody fragments thereof of the presentinvention.

In order to detect cells expressing the polypeptide, a knownimmunodetection can be used, and preferably immunoprecipitation,fluorescent cell staining or immunohistochemical staining etc. can beused. Furthermore, a fluorescent antibody staining method etc. usingFMAT8100HTS system (Applied Biosystem) can be used.

In the present invention, a living sample for detecting or measuringB7-H3 is not particularly limited, so as long as it has a possibility ofincluding cells expressing B7-H3, such as tissue cells, blood, plasma,serum, pancreatic fluid, urine, feces, tissue fluid or culture fluid.

The diagnostic reagent containing the monoclonal antibody or theantibody fragment thereof of the present invention may further contain areagent for performing antigen-antibody reaction or a reagent fordetecting the reaction, depending on desired diagnostic method. Thereagent for performing antigen-antibody reaction includes buffers,salts, and the like. The reagent for detection includes reagentscommonly used in immunodetection or measurement, such as labeledsecondary antibodies that recognize the monoclonal antibodies, antibodyfragments or conjugates thereof, substrates corresponding to the labels,etc.

The present invention also relates to a method of treating diseasesrelated to human B7-H3 positive cells, particularly in the treatment ofcancer and inflammation.

2. Examples

The present invention is further described below in conjunction with theexample, however the scope of the present invention is not limitedthereto. In the examples of the present invention, where specificconditions are not described, the experiments are generally conductedunder conventional conditions as described in Cold Spring HarborAntibody Technology Laboratory Manual, Molecular Cloning Manual, orunder conditions recommended by the manufacturer of raw material orproducts. Where the source of the reagents is not specifically given,the reagents are commercially available conventional reagents.

Example 1. Preparation of B7-H3 Antigen and Protein for Detection

Design of B7-H3 Antigen

The human B7-H3 sequence as shown in SEQ ID NO: 1 was used as thetemplate for B7-H3 of the present invention, and the amino acid sequenceof the antigen and protein for detection involved in the presentinvention were designed. Unless otherwise specified, the following B7-H3antigen is human B7-H3.

Human B7-H3 full-length amino acid sequence (SEQ ID NO: 1):

MLRRRGSPGMGVHVGAALGALWFCLTGA LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGAN

GQEIA

Note:

The double-underlined portion is the signal peptide (Signal peptide:1-28);

The underlined portion is the B7-H3 extracellular domain (Extracellulardomain: 29-466), wherein 29-139 is Ig-like V-type 1 Domain, and 145-238is Ig-like C2-type 1 Domain; 243-357 is Ig-like V-type 2 Domain, 363-456is Ig-like C2-type 2 Domain;

The dot-lined portion is the transmembrane domain portion (Transmembranedomain: 467-487);

The italic portion is the intracellular domain (Cytoplasmic domain:488-534).

Murine B7-H3 full-length amino acid sequence (SEQ ID NO: 2)

MLRGWGGPSVGVCVRTALGVLCLCLTGA VEVQVSEDPVVALVDTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRLQRVRVTDEGSYTCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCSSYQGYPEAEVFWKDGQGVPLTGNVTTSQMANERGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQ

DGDGEGSKTALRPLKPSENKEDDGQEIA

Note:

The double-underlined portion is the signal peptide (Signal peptide:1-28);

The underlined portion is the B7-H3 extracellular domain (Extracellulardomain: 29-248), wherein 29-139 is Ig-like V-type Domain, and 145-238 isIg-like C2-type Domain;

The dot-lined portion is the transmembrane domain portion (Transmembranedomain: 249-269);

The italic portion is the intracellular domain (Cytoplasmic domain:270-316).

The human B7-H3 antigen (SEQ ID NO: 3) used for screening and detectionis a commercial product (R&D cat #1949-B3-050/CF, abbreviated as2Ig-B7-H3), and the sequence is as follows:

LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTG-HHHHHH

Note: The underlined portion is the B7-H3 extracellular region; theitalic portion is the His-tag marker.

The human B7-H3 antigen (SEQ ID NO: 4) used for detection is acommercial product (SinoBiological cat #11188-H08H, abbreviated as4Ig-B7-H3), and the sequence is as follows:

LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPM T-HHHHHH

Note: The underlined portion is the B7-H3 extracellular region; theitalic portion is the His-tag marker.

The murine B7-H3 antigen (SEQ ID NO: 5) used for screening and detectionis a commercial product (R&D cat #1397-B3-050/CF), and the sequence isas follows:

VEVQVSEDPVVALVDTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRLQRVRVTDEGSYTCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCSSYQGYPEAEVFWKDGQGVPLTGNVTTSQMANERGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPLTF-HHHHHH

Note: The underlined portion is the B7-H3 extracellular region; theitalic portion is the His-tag marker.

Example 2. Screening Positive Sequence(s) for Specific Binding to HumanB7-H3

B cells were isolated from human peripheral blood mononuclear cells(PBMCs), spleen, and lymph node tissues, and RNA was extracted toconstruct naive scFv phage antibody library (capacity 3.2×10¹⁰). Theconstructed naive scFv phage antibody library was packaged to form phageparticles, and then subjected to panning by liquid phase method. Thephage was bound to the biotinylated B7-H3 in liquid phase, and then wasseparated by streptavidin magnetic beads. In order to obtain positivesequence(s) binding to human B7-H3 (R&D cat #1949-B3-050/CF),biotinylated human B7-H3 was used for panning, and 500 monoclonalcolonies were picked and packaged into phage scFv antibodies for phageELISA testing. The binding activity of monoclonal phage to human B7-H3(R&D cat #1949-B3-050/CF) and murine B7-H3 (R&D cat #1397-B3-050/CF)were tested separately: 1 μg/ml human B7-H3 or murine B7-H3 and 1% BSAwere coated on ELISA plate, phage supernatant diluted at 1:1 withblocking buffer was added, and detected with anti-M13 HRP; the cloneswith ELISA OD450 value of greater than 0.5, and with ratios of ELISAOD450 values for binding with human or murine B7-H3 to ELISA OD450 valuefor binding with 1% BSA greater than 2.0 were selected, and 9 cloneswere obtained.

Example 3. Construction of Full Length Monoclonal Antibodies

Full length antibodies were constructed for these 9 clones obtained byphage library screening, and then two antibodies (h1702 and h1703,respectively) were confirmed to have strong affinity by ELISA bindingassay. The process of constructing full length monoclonal antibody wasas follows:

Based on the scFv antibody sequence(s) obtained by phase screening,primers were designed to construct the VH/VK/VL gene fragment of eachsingle-chain antibody sequence by PCR. The heavy and light chainvariable regions of h1702 and h1703 were obtained.

>h1702 heavy chain variable region sequence SEQ ID NO: 6QVQLVQSGGGVVQPGTSLRLSCAAS GFIFSSSA MHWVRQAPGKGLEWV AV ISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARSARLYASFDYWGQGALVTVSS >h1702 light chain variable region sequence SEQ ID NO: 7QTVVTQEPSFSVSPGGTVTLTCGLS SGSVSTSHY PSWYQQTPGQAPRM LIY NTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYC AIHVDR DIWVFGGGTHKLTVL >h1703 heavy chain variable region sequence SEQ ID NO: 8QVQLQESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWV SA ISGSGGSTYYADSVKGRYTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGVGPVHALDVWGQGTTVTVSS >h1703 light chain variable region sequence SEQ ID NO: 9DIRLTQSPSSLSASVGDRVTITCRAS QSISTY LNWYQQKPGKAPILLI N AVSGLQSGVPSRFSGSGSGTHFTLTITSLQPEDFATYYC QQSYSTPM WT FGQGTKVEIK

Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics insequence are FR sequences, and the CDR sequences are underlined.

The CDR sequences in the light chain and heavy chain of each antibodyare shown in Table 1.

TABLE 1 CDR regions of each heavy and light chain Anti- body HC LC 1702HCDR1 GFIFSSSA LCDR1 SGSVSTSHY SEQ ID NO: 10 SEQ ID NO: 13 HCDR2ISYDGSNK LCDR2 NTN SEQ ID NO: 11 SEQ ID NO: 14 HCDR3 ARSARLYASFDY LCDR3AIHVDRDIWV SEQ ID NO: 12 SEQ ID NO: 15 1703 HCDR1 GFTFSSYA LCDR1 QSISTYSEQ ID NO: 16 SEQ ID NO: 19 HCDR2 ISGSGGST LCDR2 AVS SEQ ID NO: 17SEQ ID NO: 20 HCDR3 AKGVGPVHALDV LCDR3 QQSYSTPMWT SEQ ID NO: 18SEQ ID NO: 21

The antibody variable region was then homologously recombined with theconstant region gene (CH1-FC/CL) fragment to construct the completeantibody VH-CH1-FC/VK-CL/VL-CL.

The constructed complete antibodies h1702-IgG1, h1703-IgG1 sequences areas follows:

h1702-IgG1: h1702-IgG1 heavy chain amino acid sequence: (SEQ ID NO: 22)QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKh1702 light chain amino acid sequence: Lamda (SEQ ID NO: 23)QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECSh1703-IgG1: h1703-IgG1 heavy chain amino acid sequence: (SEQ ID NO: 24)QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRYTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVGPVHALDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKh1703 light chain amino acid sequence: Kappa (SEQ ID NO: 25)DIRLTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPILLINAVSGLQSGVPSRFSGSGSGTHFTLTITSLQPEDFATYYCQQSYSTPMWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

In order to further improve the stability of the antibody, the aminoacids of the hi 702 light chain sequence were mutated, wherein thespecific mutation involves that the first amino acid residue Q atN-terminus of the light chain was replaced by D, the first amino acidresidue S at C-terminus was deleted, so as to obtain a more stable anduniform monoclonal antibody.

h1702-1 light chain amino acid sequence with mutation modification: (SEQID NO: 26)

DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTEC

Example 4. Expression and Purification of Fully Human Antibodies

The plasmids expressing the light and heavy chain of the antibodyrespectively were transfected into HEK293E cells at a ratio of 1.5:1.The cell culture supernatant was collected 6 days later, and the celldebris was removed by high-speed centrifugation, and purification wasperformed by a Protein A column. The column was washed with PBS untilthe A280 reading dropped to the baseline. The target protein was elutedwith an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1 MTris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentratedand further purified by gel chromatography Superdex 200 (GE) which wasequilibrated by PBS to remove the aggregate. The monomer peak wascollected and aliquoted for use.

The performance and beneficial effect of the antibodies of the presentinvention was tested by the following test methods:

Test Example 1. ELISA Binding Assay

To test the binding ability of the screened B7-H3 antibodies todifferent forms of human B7-H3 in vitro, human 2Ig-B7-H3 (Cat.#1949-B3-050/CF, R&D) and human 4Ig-B7-H3 (Cat #11188-H08H, SinoBiological) were used for binding assays in vitro.

Human B7-H3 protein (2Ig/4Ig) was diluted to a concentration of 1 μg/mlwith phosphate-buffered saline (PBS) buffer, pH 7.4 (Sigma,P4417-100TAB), and added to a 96-well Elisa plate at a volume of 100μl/well (Corning, CLS3590-100 EA), and placed at 4° C. overnight, for16-20 hours. After discarding the liquid, 5% skim milk (GuangMing skimmilk powder) blocking solution diluted with PBST buffer (pH 7.4 PBScontaining 0.05% Tween-20) was added at 120 μl/well, and the mixture wasincubated at 37° C. for 2 hours to block. At the end of the blocking,the blocking solution was discarded, and the plate was washed 4 timeswith PBST buffer. Then, 100 μl/well of the corresponding B7-H3antibodies with series dilution was added. The initial concentration was1 μM, then diluted with PBST buffer to 8 gradients, and incubated at 37°C. for 1 hour in the incubator. After incubation, the reaction solutionin the plate was discarded, and the plate was washed 4 times with PB ST.HRP-labeled goat anti-Human IgG Fcγ fragment specific secondary antibody(Jackson Immuno Research, 109-005-008) diluted with PBST (1:4000) wasadded at 100 nl/well, and incubated for 1 hour at 37° C. After washingthe plate 4 times with PBST, TMB chromogenic substrate (KPL, 52-00-03)was added at 100 μl/well, incubated for 3-5 minutes (min) at roomtemperature, and 1 M H2504 was added at 100 μl/well to stop thereaction. The absorbance value was read using NOVOStar microplate readerat 450 nm, and the EC50 value for the binding between the antibody andthe antigen was calculated. The results are shown in Table 2, FIG. 1 andFIG. 2.

TABLE 2 The binding ability of different antibodies to human 2Ig-B7-H3and 4Ig-B7-H3 antigen EC50 human 2Ig-B7-H3(nM) human 4Ig-B7-H3(nM) h17020.11 0.16 h1703 10.28 2.10

The results show that h1702 and h1703 have significant binding abilityto both human 2Ig-B7-H3 and 4Ig-B7-H3, and h1702 has stronger bindingability.

Test Example 2. Cross-Binding Assay to B7-H3 Derived from DifferentSpecies

To test the binding ability of the screened B7-H3 antibodies to B7-H3derived from different species in vitro, murine B7-H3 (Cat.#1397-B3-050/CF, R&D) was used for binding assays in vitro.

B7-H3 protein derived from different species (mouse B7-H3) was dilutedto a concentration of 1 μg/ml with PBS buffer, pH 7.4 (Sigma,P4417-100TAB), added to a 96-well microtiter plate at a volume of 100μl/well (Corning, CLS3590-100 EA), and placed at 4° C. overnight for16-20 hours. After discarding the liquid, 5% skim milk (GuangMing skimmilk powder) blocking solution diluted with PBST buffer (pH 7.4 PBScontaining 0.05% Tween-20) was added at 120 μl/well, and the mixture wasincubated at 37° C. for 2 hours to block. At the end of the blocking,the blocking solution was discarded, and the plate was washed 4 timeswith PBST buffer. Then, 100 μl/well of the corresponding B7-H3antibodies at an initial concentration of 1 μM were added, diluted withPBST buffer to 8 gradients, and incubated at 37° C. for 1 hour in theincubator. After incubation, the reaction solution in the plate wasdiscarded, and the plate was washed 4 times with PBST, and HRP-labeledgoat anti-Human IgG Fcγ fragment specific secondary antibody (JacksonImmuno Research, 109-005-008) diluted with PBST (1:4000) was added at100 μl/well, incubated for 1 hour at 37° C. After washing the plate 4times with PBST, TMB chromogenic substrate (KPL, 52-00-03) was added at100 μl/well, incubated for 3-5 min at room temperature, and 1 M H₂SO₄was added at 100 μl/well to stop the reaction, the absorbance value wasread using NOVOStar microplate reader at 450 nm, the EC50 value for thebinding between the antibody and the antigen was calculated (the resultsare shown in Table 3 and FIG. 3).

TABLE 3 The binding ability of different antibodies to murine B7-H3antigen EC50 murine B7-H3 (nM) h1702 18.12 h1703 86.68

The results shown that the binding ability of h1702 and h1703 to murineB7-H3 was weak, indicating that two monoclonal antibodies specificallybind to human B7-H3.

Test Example 3. Biacore Test for Antibody Affinity

The reaction affinity of anti-B7-H3 antibodies to human and murine B7-H3was determined using a Biacore, GE instrument.

A biosensor chip Protein A (Cat. #29127556, GE) was used to affinitycapture a certain amount of antibody to be tested. A series diluted ofhuman 2Ig-B7-H3 antigen (Cat. #1949-B3-050/CF, R&D), human 4Ig-B7-H3antigen (Cat. #11188-H08H, Sino Biological) or murine B7-H3 antigen(Cat. #1397-B3-050/CF, R&D) was flowed through the surface of the chip.Real-time reaction signal was detected by using Biacore instrument(Biacore T200, GE), in order to obtain association and dissociationcurves. After completion of each cycle of dissociation, the biochip waswashed and regenerated with glycine-hydrochloric acid regenerationsolution (pH 1.5) (Cat. # BR-1003-54, GE). The buffer used in theexperiment was HBS-EP buffer solution (pH 7.4) (Cat. # BR-1001-88, GE).

The experimental data was fitted with BIA evaluation version 4.1 GEsoftware in a (1:1) Langmuir model to obtain affinity values. Theexperimental results are shown in Tables 4-6.

TABLE 4 The reaction affinity of different antibodies to human 2Ig-B7-H3antigen Antibody Antigen Affinity (M) h1702 human 2Ig-B7-H3 7.97E−7h1703 4.48E−7

TABLE 5 The reaction affinity of different antibodies to human 4Ig-B7-H3antigen Antibody Antigen Affinity (M) h1702 human 4Ig-B7-H3 8.55E−9h1703 —

TABLE 6 The reaction affinity of different antibodies to murine B7-H3antigen Antibody Antigen Affinity (M) h1702 murine B7-H3 1.47E−6 h17035.02E−7

The affinity results of Biacore test show that h1702 has strong affinityto human 4Ig-B7-H3, reaching a level at nM, whereas the affinity tohuman 2Ig-B7-H3 or murine B7-H3 is relatively weaker; h1703 has weakeraffinity to human 4Ig-B7-H3, human 2Ig-B7-H3 and murine B7-H3.

Test Example 4. In Vitro Cell Binding Assay

In this experiment, the binding of antibody was evaluated based on theintensity of fluorescence signal of the antibody bond on the cellsurface. After incubating 10 μg of primary antibody with 2×10⁵ U87MGcells on ice for 30 minutes, excess antibody was removed by washing. Thecells were incubated with APC anti-human IgG Fc (Biolegend, 409306) for30 minutes at room temperature, and after removing excess antibody, thefluorescence signal on the cell surface was read using BD Verse (resultswere shown in FIG. 4). The results indicate that h1702 specificallybinds to U87MG tumor cells which overexpress B7-H3.

Test Example 5. In Vitro Endocytosis Test

In this experiment, the endocytosis effect of the antibody was evaluatedbased on the intensity of fluorescence signal which was determined ofthe internalized antibody. The B7-H3 antibody and APC anti-human IgG Fc(Biolegend, 409306) were mixed at a molar ratio of 1:2 and incubated onice for 15 minutes. After incubating the antibody mixture with 2×10⁵U87MG cells on ice for 30 minutes, excess antibody was removed bywashing, and then the cells were transferred to a 37° C. prewarmedmedium, and incubated at 37° C. for 0, 15, 30, 60 and 120 minutesrespectively. The cells were centrifuged and resuspended in the antibodyelution buffer to get rid of antibodies. After incubating for 7 minutesat room temperature, the antibody elution buffer was removed by washing,and the intracellular fluorescence signal was read using BD Verse(results shown in FIG. 5). The results show that both h1702 and h1703were efficiently endocytosed into cells after binding to U87MG cells.

Test Example 6. T1/2 Evaluation on SD Rats

4 SD rats (purchased from JSJ Experimental Animal Co., Ltd.), 2 malesand 2 females, were maintained in light/dark cycle adjusted at 12/12hours, with constant temperature of 24±3° C., humidity of 50-60%, andfree access to food and water. On the day of the experiment, SD ratswere injected with the test agent into tail vein at a dose of 3 mg/kgand an injection volume of 5 ml/kg.

The time for blood collection: On the first day of administration, bloodwas taken from ocular fundus vein at 5 min, 8 h, 24 h, 2 days, 3 days, 5days, 8 days, and 15 days after administration, 200 μL each time(equivalent to 100 μL of serum). The collected blood samples wereallowed to incubateat room temperature for half an hour untilcoagulation, and then centrifuged at 10,000×g for 10 minutes at 4° C.The supernatant was collected and immediately stored at −80° C. TheB7-H3 antibody concentration in the serum was measured by ELISA, and PKanalysis was performed. The results are shown in Table 7.

TABLE 7 T_(1/2) of B7-H3 antibody in SD rats Tested agent Route ofadministration T_(1/2) (mean ± SD, h) h1702 IV (3 mg/kg) 185 ± 17

The results show that the half-life of the antibody of the presentinvention in rats was approximately 185 h (7.7 days).

Test Example 7. Physical Stability of B7-H3 Antibodies

Differential scanning calorimetry (DSC) was used to detect the thermalstability of different antibodies, and the thermal stability indifferent buffer systems under different pH conditions was compared.Exemplary buffer systems corresponding to different pHs were 10 mM PB(pH 7) and 10 mM Acetate (pH 5.2). The sample was dissolved in thecorresponding buffer, and the sample concentration was controlled atabout 1 mg/ml. Detection was performed using MicroCal* VP-Capillary DSC(Malvern). Before detection, each sample and the blank buffer weredegassed by a vacuum degasser for 1 to 2 minutes. 400 μl of sample orblank buffer were added to each well of the sample plate (the load ofinstrument is 300 μl). The last two pairs of well-plates wererespectively added with 14% Decon 90 and ddH₂O for cleaning. After thesample plate was loaded, a plastic soft cover was placed. The scanningtemperature started from 25° C. and ended at 100° C. The scanning ratewas 60° C./h. The results are shown in Table 8. In several test systems,both h1702 and h1703 showed good thermal stability.

TABLE 8 DSC results of different antibodies Sample Buffer Tm-onset (°C.) TM (° C.) h1702 pH 7.0 65.82 77.51 pH 5.2 65.59 78.97 h1703 pH 7.061.33 73.19 pH 5.2 60.65 75.71

The purity of the sample was monitored by SEC-HPLC to investigate thestability under a certain concentration condition. As an example ofcondition, the sample concentration was controlled at about 40-50 mg/ml.The stability of different antibodies was compared in PBS (pH 7.4)system and pH 5.2 acetic acid/sucrose system at 4° C., 30° C., 40° C.for one month of storage. The purity of the antibody was examined usingXbridge protein BEH SEC 200A (Waters) HPLC column. After one month ofinvestigation, both h1702 and h1703 showed good stability. The resultsare shown in Table 9.

TABLE 9 Stability results for different antibodies 4° C./month/ 30°C./month/ 40° C./month/ Sample purity purity purity h1702/acetic 99.25%98.68% 97.85% acid h1702/PBS 99.21% 98.07% 96.34% h1703/acetic 99.31%99.04% 98.38% acid h1703/PBS 99.18% 98.56% 96.99%

The results show that both h1702 and h1703 show excellent stability inboth acetic acid and PBS buffers.

Test Example 8. Chemical Stability of B7-H3 Antibodies

Chemical modification after antibody preparation is one of the commonreasons leading to the product stability problem, especially the highdegree of deamination, oxidation or isomerization modification at someamino acids in the CDR region. Those modifications should be avoided orreduced. 500 μg of the antibodies to be tested was dissolved in 500 μlof PBS pH 7.4, and subjected to a water bath at 40° C. Samples weretaken at day 0, 10, and 20, respectively, for enzymatic hydrolysisexperiments. 100 μg samples were taken at different time points anddissolved in 100 μl solution of 0.2 M His-HCl, 8 M Gua-HCl, pH 6.0; 3 μlof 0.1 g/mL DTT was added, and subjected to water bath at 50° C. for 1hour; Afterward, the samples were ultra-filtered twice with solution of0.02 M His-HCl, pH 6.0, and 3 μL of 0.25 mg/mL trypsin was added. Themixture was hydrolyzed overnight at 37° C. in water bath. Potentialmodification sites were analyzed by mass spectrometry (the results areshown in Table 10) using Agilent 6530 Q-TOF. The results show that h1702and h1703 described in the present invention have no significantlyincreased trend towards deamidation, oxidation or heterogeneity,indicating that the molecules have excellent chemical stability.

TABLE 10 Chemical stability of different antibodies Sample LC/HCposition/modification D 0 D 10 D 20 h1702 LC M48/oxidation 2.82% 2.9%2.83% HC M34/oxidation 3.52% 3.46% 3.38% M83/oxidation 0.98% 1.01% 0.01%h1703 HC M34/oxidation 1.93% 2.62% 2.16% M83/oxidation 1.96% 2.62% 2.8%

Test Example 9. Stability of h1702-1 Antibody

The lambda type light chain has one more amino acid S at the C-terminusthan kappa type light chain, and the steric hindrance of the S may be afactor causing instability of interchain disulfide bond between lightand heavy chain. The terminal amino acid S can be knocked out bymolecular cloning, and the stability of the antibody under alkalinecondition would be remarkably improved.

When the first amino acid at the N-terminus of the light chain of thenaked antibody is Q, partial cyclization would also occur in theantibody, which leads to an increase in charge heterogeneity of thesample, affecting the stability of formulation and product. The firstamino acid Q at N-terminus was mutated to D by molecular cloning, toeliminate the incomplete cyclization and significantly improve theantibody stability. The above modification did not significantly affectthe affinity of the engineered antibody to its antigen.

The stability of h1702 and h1702-1 was tested by size-exclusionchromatography (SEC), non-reducing CE-SDS analysis method (pH 9.0) andIEX analysis method.

SEC detection: Waters e2695 chromatograph and Xbridge BEH 200A SECcolumn were used. 50 μg of antibody was loaded, and the elution wasperformed using PBS mobile phase in constant gradient.

CE-SDS NR Method:

Samples were processed using the Beckman SDS-MW Analysis Kit. A buffersolution was added to 100 μg tested antibody was denatured by heating insample buffer as described in manned protocol. Data was collected usingPA800 capillary electrophoresis apparatus.

IEX Method:

Waters Acquity H-Class chromatograph and Thermo MAbPac SCX-10 columnwere used. 50 μg of tested antibody was loaded, and a linear gradientwas applied, using CX-1 pH Gradient Buffer Kit as the mobile phase;ultraviolet signal at a wavelength of 280 nm was collected.

TABLE 11 Comparison of stability of h1702 and h1702-1 SEC CE-SDS (pH9.0) IEX h1702 100% 71.21% 40.5% h1702-l 100% 94.67% 86.21%

Although the invention has been described in detail with the figures andspecific embodiments for the purpose of a clear understanding, thedescription and embodiments should not be interpreted as limiting thescope of the present invention. All public contents of literatures andpatents cited herein are expressly incorporated by reference in theirentirety.

1. A monoclonal anti-B7-H3 antibody or antigen-binding fragment thereof,which binds to human B7-H3, comprising: (i) an antibody heavy chainvariable region comprising HCDR1, HCDR2, and HCDR3 having the amino acidsequences of SEQ ID NOs: 10, 11 and 12, respectively; and an antibodylight chain variable region comprising LCDR1, LCDR2, and LCDR3 havingthe amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively; or(ii) an antibody heavy chain variable region comprising HCDR1, HCDR2,and HCDR3 having the amino acid sequences of SEQ ID NOs: 16, 17 and 18,respectively; and an antibody light chain variable region comprisingLCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs:19, 20 and 21, respectively.
 2. The monoclonal anti-B7-H3 antibody orantigen-binding fragment thereof according to claim 1, wherein themonoclonal antibody is a recombinant antibody.
 3. The monoclonalanti-B7-H3 antibody or antigen-binding fragment thereof according toclaim 2, wherein the monoclonal antibody is a human recombinant antibodyor antigen-binding fragment thereof.
 4. The monoclonal anti-B7-H3antibody or antigen-binding fragment thereof according to claim 3,wherein the framework (FR) sequences of the light chain and heavy chainvariable regions of the human recombinant antibody are derived from ahuman germline light chain and heavy chain, respectively, or mutantsequences thereof.
 5. The monoclonal anti-B7-H3 antibody orantigen-binding fragment thereof according to claim 4, wherein the humanrecombinant antibody comprises a heavy chain variable region having theamino acid sequence of SEQ ID NO: 6 or 8 or a variant thereof; whereinthe variant has a deletion, substitution or addition of 1-10 amino acidsin the heavy chain variable region of SEQ ID NO: 6 or
 8. 6. Themonoclonal anti-B7-H3 antibody or antigen-binding fragment thereofaccording to claim 4, wherein the human recombinant antibody comprises alight chain variable region having the amino acid sequence of SEQ ID NO:7 or 9 or a variant thereof; wherein the variant has a deletion,substitution or addition of 1-10 amino acids in the light chain variableregion of SEQ ID NO: 7 or
 9. 7. The monoclonal anti-B7-H3 antibody orantigen-binding fragment thereof of claim 1, wherein the anti-B7-H3antibody further comprises a human antibody constant region, and theanti-B7-H3 antibody is a full-length antibody having the heavy chain andlight chain amino acid sequences as shown in SEQ ID NOs: 22 and 23,respectively, or a full-length antibody having the heavy chain and lightchain amino acid sequences as shown in SEQ ID NOs: 22 and 26,respectively, or a full-length antibody having the heavy chain and lightchain amino acid sequences as shown in SEQ ID NOs: 24 and 25,respectively.
 8. The monoclonal anti-B7-H3 antibody or antigen-bindingfragment thereof of claim 1, wherein the antigen-binding fragment isselected from the group consisting of a Fab, Fab′, F(ab′)2 andsingle-chain antibody (scFv), dimerized V region (diabody),disulfide-stabilized V region (dsFv) and CDR-containing peptide. 9.(canceled)
 10. A pharmaceutical composition comprising a therapeuticallyeffective amount of the monoclonal anti-B7-H3 antibody orantigen-binding fragment thereof of claim 1, and one or morepharmaceutically acceptable carriers, diluents or excipients.
 11. Anucleic acid molecule; encoding the monoclonal anti-B7-H3 antibody orantigen-binding fragment thereof of claim
 1. 12. A recombinant vector,comprising the nucleic acid molecule of claim
 11. 13. A host celltransformed with the recombinant vector according to claim 12, whereinthe host cell is selected from the group consisting of a prokaryoticcell and eukaryotic cell.
 14. A method for producing the monoclonalanti-B7-H3 antibody or antigen-binding fragment thereof according toclaim 1, wherein the method comprises: cultivating a host celltransformed with a recombinant vector comprising a nucleic acidmolecule; encoding the monoclonal anti-B7-H3 antibody or antigen-bindingfragment thereof in a culture to form and accumulate the monoclonalanti-B7-H3 antibody or antigen-binding fragment thereof, and recoveringthe accumulated monoclonal anti-B7-H3 antibody or antigen-bindingfragment thereof from the culture.
 15. A method for immunologicallydetecting or measuring B7-H3, wherein the method comprises detecting theB7-H3 by using the monoclonal anti-B7-H3 antibody or antigen-bindingfragment thereof of claim
 1. 16-17. (canceled)
 18. A method fordiagnosing a disease related to a human B7-H3 positive cell, wherein themethod comprises detecting or measuring the B7-H3 or B7-H3 positive cellby using the monoclonal anti-B7-H3 antibody or antigen-binding fragmentthereof of claim
 1. 19. (canceled)
 20. A method for treating a diseaserelated to a B7-H3 positive cell, wherein the method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the B7-H3 antibody or antigen-binding fragment thereof ofclaim
 1. 21. A monoclonal anti-B7-H3 antibody or antigen-bindingfragment thereof, comprising a heavy chain variable region and a lightchain variable region having the amino acid sequences of SEQ ID NOs: 6and 7, respectively; or the amino acid sequences of SEQ ID NOs: 8 and 9,respectively.
 22. A pharmaceutical composition comprising the monoclonalanti-B7-H3 antibody or antigen-binding fragment thereof of claim 21, andone or more pharmaceutically acceptable carriers, diluents orexcipients.
 23. A method of treating a disease related to a B7-H3positive cell in a subject in need thereof, comprising administering tothe subject the pharmaceutical composition of claim
 10. 24. A method oftreating a disease related to a B7-H3 positive cell in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of the pharmaceutical composition of claim 22.